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Template:Team:ATOMS-Turkiye/Lab:Protocols:Ellman
From 2013.igem.org
Contents |
Ellman’s Assay
Procedure for Quantitating Sulfhydryl Groups Using a Cysteine Standard
A. Material Preparation
• Reaction Buffer: 0.1M sodium phosphate, pH 8.0, containing 1mM EDTA • Cysteine Hydrochloride Monohydrate: M.W. = 175.6, Product No. 44889 (see Related Thermo Scientific Products) • Ellman’s Reagent Solution: Dissolve 4mg Ellman’s Reagent in 1mL of Reaction Buffer.
B. Procedure
1. Prepare a set of cysteine standards by dissolving Cysteine Hydrochloride Monohydrate at the following concentrations in Reaction Buffer:
- Standard Volume of Reaction Buffer Amount of Cysteine (M.W. = 175.6) Final Concentration A ell100mL 26.34mg 1.5mM B 5mL 25mL of Standard A 1.25mM C 10mL 20mL of Standard A 1.0mM D 15mL 15mL of Standard A 0.75mM E 20mL 10mL of Standard A 0.5mM F 25mL 5mL of Standard A 0.25mM G 30mL 0ml 0.0mM (Blank)
2. Prepare a set of test tubes, each containing 50µL of Ellman’s Reagent Solution and 2.5mL of Reaction Buffer. 3. Add 250µL of each standard or unknown to the separate test tubes prepared in step 2. Note: For the unknown(s), make dilutions so that the 250µL sample applied to the assay reaction has a sulfhydryl concentration in the working range of the standard curve (0.1-1.0mM is ideal). 4. Mix and incubate at room temperature for 15 minutes. 5. Measure absorbance at 412nm. 6. Plot the values obtained for the standards to generate a standard curve. Determine the experimental sample concentrations from this curve. Note: The most accurate results are obtained from the linear portion of the standard curve; i.e, the portion yielding an r2 value equal to 1.0. One or more of the high standards may exceed the linear range.
Procedure for Quantitating Sulfhydryl Groups Based on Molar Absorptivity
A. Material Preparation
• Reaction Buffer: 0.1M sodium phosphate, pH 8.0, containing 1mM EDTA • Ellman’s Reagent Solution: Dissolve 4mg Ellman’s Reagent in 1mL of Reaction Buffer.
B. Measure Absorbance
1. For each unknown sample to be tested, prepare a tube containing 50µL of Ellman’s Reagent Solution and 2.5mL of Reaction Buffer. 2. Add 250µL of each unknown to the separate test tubes prepared in step 1. As a blank, add 250µL of Reaction Buffer to a separate test tube prepared in Step 1. Note: For the unknown(s), make dilutions so that the 250µL sample applied to the assay reaction has a sulfhydryl concentration less than 1.0mM. Concentrations exceeding 1mM free sulfhydryl will result in high absorbance values and less accurate estimation of the concentration based on the extinction coefficient. 3. Mix and incubate at room temperature for 15 minutes. 4. With a spectrophotometer set to 412nm, zero the instrument on the blank and then measure absorbance of each sample. 5. Calculate the amount and concentration of sulfhydryls in the sample from the molar extinction coefficient of TNB (14,150M-1cm-1), as exemplified in Section C.
